Early detection of mild cognitive impairment (MCI) symptoms is an important step to its diagnosis and intervention. We developed a new screening test called “Efficient Online MCI Screening System” (EOmciSS) for use in community-dwelling older adults. It is a self-paced cognitive test to be completed within 10 minutes on tablets or smartphones in homes or care centers for older adults.This study aims to test the validity of EOmciSS for identifying community-dwelling older adults with MCI risks.Participants (N=827) completed EOmciSS and other screening tests for MCI. The psychometric properties tested were “subscale item difficulty,” “discriminative index,” “internal consistency,” and “construct validity.” We also tested between-group discrimination using the cross-validation method in an MCI group and a normal cognitive function (NCF) group.P<.001; CD-Acc1, P=.07; CD-Acc2, P=.06; CD-Acc3, P<.001; TM-Acc4, P=.07; DR-Acc1, P=.03; RS, P=.06; DR-RT1, P=.02; and DR-RT2, P=.05) that were significant predictors for an MCI classification versus NCF classification. Depressive symptoms were identified as significant factors (P<.001) influencing the performance of participants, and were an integral part of our test system. Age (P=.15), number of years of education (P=.18), and proficiency in using an electronic device (P=.39) did not significantly influence the scores nor classification of participants. Application of the MCI/NCF cutoff score (7.90 out of 9.67) to the validation data set yielded an area under the curve of 0.912 . The sensitivity was 84.9%, specificity was 85.1%, and the Youden index was 0.70.A total of 3 accuracy factors and 1 reaction time factor explained the structure of the 20 item factors. The difficulty level of accuracy factors  was 0.63-0.99, whereas that of the reaction time factor was 0.77-0.95. The discriminative index of the medium-to-high-difficulty item factors was 0.39-0.97. The internal consistency (Cronbach α) ranged from .41 (for few item factors) to .96. The training data set contained 9 item factors . Depressive symptoms should be an integral part in future MCI screening systems because they influence the test performance and, hence, MCI risk.Chinese Clinical Trial Registry ChiCTR2000039411; http://www.chictr.org.cn/showprojen.aspx?proj=62903 Approximately 50 million people worldwide suffer from dementia, and this number is expected to increase to 152 million by 2050 . StudiesEarly detection of MCI symptoms in the community-dwelling older population is the first step of this endeavor . StudiesScreening tests can be in various forms: self-evaluation, evaluation by an examiner, or evaluation by a caregiver. Self-rating tests rely on self-reporting by the individual , and an Performance tests involve the administration and scoring of the test by a medical professional. An example is the Montreal Cognitive Assessment (MoCA). The internal consistency of the MoCA is .83 (Cronbach α) . A systeThe General Practitioner Assessment of Cognition (GPCOG) uses the caregiver as the informant in addition to professional scoring. The internal consistency of the GPCOG has been reported to be .84 (Cronbach α), with a sensitivity of 82% and specificity of 83% . The GPCApart from paper-and-pencil tests, computerized screening tests or digital screening tests take advantage of information technology and the portability of service delivery ,20. For Given the aforesaid unmet needs and shortcomings, we developed a new screening test called “Efficient Online MCI Screening System” (EOmciSS) for use by community-dwelling older adults. First, we selected test items of relatively high sensitivity and high specificity for MCI. “cube copying” (CC) and “clock drawing” (CD) have shown a sensitivity of 65.6% and 57.1% and specificity of 53.3% and 70.0%, respectively, for individuals with MCI versus individuals with NCF -29. The EOmciSS has certain features that cater to the needs and self-paced operations of older users. For example, test instructions are delivered audially and visually simultaneously. Cross-audiovisual presentations can enhance the attention and, hence, the learning of users to understand the operation of test tasks . The locWe aimed to gather evidence of the validity of EOmciSS for identifying community-dwelling older adults with MCI risks. It is a self-paced, rapid, cognitive screening test for users that can be completed within 10 minutes on tablets or smartphones in homes or care centers. We hypothesized that the test items of EOmciSS would contribute significantly to identify individuals with a high risk of MCI, and that the sensitivity and specificity of EOmciSS would be comparable with those of other paper-and-pencil tests for MCI screening.The inclusion criteria were as follows: (1) age between 55 and 75 years; (2) community dwelling and attending an activity center regularly; (3) had received education for more than 6 years ; and (4)The exclusion criteria were taking medications  in the previous 2 weeks or a known history of (1) brain trauma, brain tumor, cerebral infarction, cerebral hemorrhage, intracranial infection, Parkinson, epilepsy, or other neurological diseases; (2) depression, anxiety, schizophrenia, or other mental illnesses; (3) abuse of alcohol or other substances.Based on convenience sampling, participants (older adults) were recruited from 32 community centers for older adults in a city in China from June 2019 to December 2020.A total of 1081 participants were recruited and completed testing. Among them, 196 were verified to have received ≤6 years of education. Another 58 participants were found to have excessive amounts of missing data. The final sample size entering the analyses was 827 .Classification of participants in the MCI group was based on the diagnostic criteria published by Petersen  and the We adopted the diagnostic criteria stipulated by the National Institute on Aging-Alzheimer’s Association  to excluResearch assistants helped participants to access EOmciSS using a tablet. Participants began to complete the test with assistance from the instructions and sample items provided by EOmciSS. Research assistants did not aid the participants at any point during the test. Each participant yielded a set of EOmciSS item scores and a machine-generated total score. After completing EOmciSS, participants were administered a series of criterion tests: MoCA, AD8, and IADL. These tests were administered by a different group of research assistants who received training on delivering the 3 tests using standardized procedures. Participants were also assessed by physicians within our research team who had the experience of diagnosing MCI in their clinical practices.There are 4 main features in the conceptualization of EOmciSS that facilitate large-scale screening in a community setting. First, the composition of the test contents takes an eclectic approach. The constructs of all the test items have been supported with sufficient evidence for differentiating individuals with MCI from individuals with NCF. These are “depressive mood,” “visual attention and flexibility,” “visuospatial and executive functions,” “memory and cognitive processing speed.” Second, the user interface is simple and allows for self-administration and the test to be completed at different times in different places. Third, the time required for most users to complete the test should be 10 minutes or less. Fourth, administration of the test and scoring are automated.EOmciSS has 2 sections delivered in sequence . The firThe cognitive test has 5 subtests that contain 20 test items . The 20 A total of 3 measures were used as the external criteria for establishing evidence of the concurrent validity of EOmciSS. The C-MoCA  containsDifferent methods were used to generate evidence on the validity of the 20 test items under EOmciSS subscales. At the test level, the structure of the 20 item factors (13 Acc + 6 RT + 1 order) was explored by confirmatory factor analysis. At item and subtest levels, “item difficulty,” “discriminative index,” and “internal consistency” were the indices. “Item difficulty” refers to the proportion of correct answers per item . “Discrit test, Spearman rank correlation test, or χ2 test. Multivariate binary logistic regression was applied to test the significance of the 20 item factors for predicting the membership of participants in the NCF group or the MCI group. The area under the curve (AUC) and Hosmer-Lemeshow goodness-of-fit test were used to describe the ability of the regression model to differentiate MCI from NCF. The regression model obtained from the training set was tested with the sample in the validation set. The β coefficients yielded became the item weights of each significant item factor for computing the total score. Predicted memberships were compared with observed memberships to derive the optimal cutoff score for MCI and NCF, as well as its sensitivity, specificity, Youden index, and receiver operating characteristic curve.As suggested by Boateng et al , known-gThe Medical Ethics Committee of the Affiliated Rehabilitation Hospital of Fujian University of Traditional Chinese Medicine  approved the study protocol . The purposes of the study were explained to all participants, who provided written informed consent.P<.001). Comparative fit index, standardized root-mean-squared residual, and root-mean-square error of approximation were 0.97, 0.007, and 0.048, respectively [A total of 20 item factors made up the performance Acc (n=14) and RT (n=6): TM-Acc , CD-Acc (#8-10), CC-Acc (#11), DR-Acc (#12-14), CC-RT (#15), CD-RT (#16), TM-RT (#17), DR-RT1 (#18), DR-RT2 (#19), and RS (#20). Confirmatory factor analysis revealed the Kaiser-Meyer-Olkin value to be 0.841, and the Bartlett test showed a significant difference , visuospatial and executive function (CD and CC), memory (DR), and cognitive processing speed (RS).The difficulty level of the items under the TM, CD+CC, DR, and RT subtests was 0.69-0.90, 0.63-0.99, 0.95-0.98, and 0.77-0.95, respectively. The corresponding discriminative index was 0.626-0.974, 0.224-0.886, 0.530-0.790, and 0.25-0.60 . The intP=.74). In the training set, the MCI subgroup was significantly older (P=.048), had fewer years of education (P<.001), and spent less time using an electronic device (P<.001) than the NCF subgroup . Participants in the NCF subgroup showed a significantly higher mean score on the MoCA (P<.001) and a lower mean score on the GDS-15 (P<.001) than those in the MCI subgroup. Significant differences were not found in the mean score for the IADL scale (P=.24).There were 402 participants with NCF and 177 participants with MCI in the training set . There wsubgroup . Other bχ21=7.52-127.75, P<.001). Participants with NCF showed faster RT on correct items than those with MCI across all item factors .Participants with NCF in the training set showed significantly higher Acc than those with MCI in all EOmciSS item factors  as the significant predictor at P<.05; 5 item factors were marginally significant predictors at P=.05-.07 (P<.001) covariate in the model. The AUC of the MCI prediction model versus the NCF prediction model was 0.908 , which was over 0.75 and suggested satisfactory discriminative power. The Hosmer-Lemeshow goodness-of-fit test also indicated good data for model fitting . Weighted scores of the 9 item factors were derived based on the β coefficients [P=.07), CD-Acc2 (P=.06), TM-Acc4 (P=.07), RS (P=.06), and DR-RT2 (P=.05), despite their marginal significance in the regression model, was to strengthen content representativeness of EOmciSS  and number of years of education (P<.001). Use of an electronic device showed marginal significance in subgroup differences (P=.07). The MCI subgroup  had significantly fewer participants reporting a history of tobacco smoking than the NCF group . Participants in the NCF subgroup showed a significantly higher mean score on the MoCA (P<.001) and a lower mean score on the GDS-15 (P=.01) than those in the MCI subgroup. Significant between-subgroup differences were not found for the mean score on the IADL scale (P=.34). With the same parameters derived from the training set, the AUC for the classification of the NCF and MCI subsets was 0.912 . The cutoff score of 7.90 yielded 84.9% sensitivity, 85.1% specificity, and Youden index of 0.70 for the validation set. The receiver operating characteristic curves of the validation set are presented in The validation set comprised 248 participants. Significant between-subgroup differences were in age ; cognitive flexibility and working memory (in TM); and verbal memory (in DR). In the TM subtest, the connection from the third-to-fourth alternative symbols contributed the most to the classification model. Besides, the speed of performances in terms of RT (in RS) and recall of memory items (in DR) significantly predicted membership of the MCI group or the NCF group. Incorporation of a “depressive mood” measure in the first section of EOmciSS is a new design for a cognitive screening test. Evidence suggests that individuals with a depressive mood are likely to have low motivation and delays in executing actions, which would pose threats to the internal validity of cognitive test performances . For EOmciSS, the impacts would be on the measures of Acc and RT. The identification of users of EOmciSS with the GDS-15 before them entering into the cognitive test section would safeguard the validity of the cognitive screening for MCI.The test construct of EOmciSS was substantiated with results from the confirmatory factor analysis. The 20 item factors were clustered satisfactorily into 3 latent Acc factors with respect to the test content of visual attention and mental flexibility (TM), visuospatial and executive function (CD and CC), memory (DR), and cognitive processing speed (RS). The Acc of the 3 CD items and 1 CC item was grouped under 1 single latent factor. For CD, the 3 Acc item factors were “placing the 12 numbers  on the face of a clock” (#CD-Acc1), “orienting the hour hand” (#CD-Acc2), and “orienting the minute hand” (#CD-Acc3). The CD item had 1 Acc item factor: “drawing the cube.” Within CD items, the “orienting the minute hand” Acc item factor was the most difficult (mean 0.85), but it was less difficult than the “draw the cube” item factor (mean 0.63). CD and CC are tests for visuospatial, planning, and executive functions, which have been reported to be effective for screening for individuals with MCI ,51. TherWe emphasized the Acc and speed of participants’ performances when designing EOmciSS. EOmciSS has made possible the capture of both performance parameters at a response level rather than at an item level. For instance, CD has 3 Acc factors and 1 RT factor. The definitions for the 4 item factors are different from those in the Digital Clock Drawing study, which quantifies the size, angle, and spacing between the clock elements, and their latencies . In thisFor the CC item, Oonuma et al  reportedThe TM items, despite their marginal significance in the prediction model, appeared to have a compatibly important role as those of CD and CC. Interestingly, among the 6 factors, the “third-to-fourth symbol connection” Acc item factor (#TM-Acc4) was the best discriminative index. The “third-to-fourth symbol connection” was the only TM item to be included in the MCI versus NCF prediction model. It yielded the largest β coefficient and, therefore, was assigned with the heaviest weight contributing to the EOmciSS score. The design of the TM item is comparable with the conventional Trail Making Test ,55. A prAbout 90% (745/827) of participants completed EOmciSS in 10 minutes. This short time is a promising factor for maximizing the number of community-dwelling older adults who complete the test. Compared with other computerized tests, the CoCoSc system used by Wong et al  had 5 suOur study had 3 main limitations. First, participants were older adults who had been educated for more than 6 years. Thus, our results may not be generalizable to those who have only a few years of education or who are illiterate. We should explore possible adaptations of test content and a user interface that is appropriate for these groups. Second, compared with other computerized screening tests, EOmciSS could be used to differentiate individuals with MCI from individuals with NCF. However, the content representativeness of EOmciSS was limited. Therefore, EOmciSS data should be interpreted as indicating the risks, but not the diagnosis, of MCI. Additional neuropsychological and medical tests are recommended for reaching a clinical diagnosis. Third, we used a cross-sectional method to gather evidence, which hampers the repeatability and stability of test results. We must investigate the robustness of the test scores and classification results by using a longitudinal design and analytic methods, as described by Lei and colleagues .EOmciSS is a new, computerized, cognitive screening test designed for use by community-dwelling older adults. The test content, user interface, and administration method enable older adults to complete the test with a smartphone or tablet within 10 minutes. The test constructs of EOmciSS can be factored into Acc and RT components, both of which contributed significantly to the ability of EOmciSS to differentiate individuals with MCI risks from those with NCF. The screening ability of EOmciSS was revealed to be the strongest among other computerized tests, with comparable test constructs and time needed for completion. EOmciSS could serve as a first-line test for identifying individuals with MCI risks in the community. Application of EOmciSS could enable rapid, inexpensive, large-scale screening among older adults. Individuals who receive positive screening results should be followed up with more comprehensive neuropsychological and medical tests before confirmation of an MCI diagnosis.


The current study aimed to develop and evaluate a sustained-release transdermal Glipizide (GLP) film to overcome its oral administration problems. Chitosan (CS)-coated deformable liposomes (DLs) were utilized to enhance the drug transdermal delivery. The formulations were characterized in terms of particle size, zeta potential, entrapment efficiency (EE%), vesicle deformability, morphology, stability, and in vitro release. Transdermal films of chosen formulations were prepared by the solvent casting technique, and an ex vivo study throughout rat skin was also performed. Moreover, a pharmacokinetics (PK) study was carried out and blood glucose levels were estimated. All the liposomes were in the nanometer range and a high EE% was obtained from DLs compared to conventional liposomes (CL). The prepared formulations showed a high stability and the DLs exhibited a high deformability compared to CL. The in vitro release study confirmed the sustained release of GLP from both CL and DL and a more pronounced sustained release of GLP was detected after coating with CS. Moreover, GLP was shown to efficiently permeate through the rat skin from transdermal films by an ex vivo permeation test. The transdermal films showed a promising PK profile in the rat as compared with oral GLP. Most importantly, GLP-CS-DL1 demonstrated a higher hypoglycemic effect, confirming the possibility of systemic action by the local topical delivery of GLP. Like other drugs among the second-generation sulfonylureas, glipizide (GLP) acts by stimulating insulin secretion from pancreatic beta-cells as well as modifying the responsiveness of insulin-sensitive tissues . GLP belSeveral attempts have been made to improve the bioavailability of oral GLP and hence patient compliance. A sustained-release system is one of the approaches used in several studies. It is likely that transdermal films are efficient for treating chronic disorders such as diabetes mellitus because they can successfully manage blood sugar with minimal side effects of oral delivery. This system may be particularly useful to control the blood glucose level and sustain the drug release. The administration of GLP by the transdermal route could overcome the problems associated with its oral route resulting in improved bioavailability ,4. To thDue to their structural similarity with the skin constituents, biocompatibility, and biodegradability, liposomes are especially interesting options for percutaneous delivery that have been used successfully to transport drugs across the skin. There have been reports on the interaction between lipids present in the stratum corneum’s outer layers and the liposome lipid bilayer, changing the structure of the upper skin. Subash et al. concluded that glimepiride-loaded liposomes showed a successful sustained-release property . Issues Deformable liposome (DL) is a highly adaptable, stress-responsive, complex aggregate that can cross barriers efficiently. In contrast to conventional liposomes, whose permeation is limited to the stratum corneum’s outer layers, DLs can be transported as complete vesicles across the skin to reach the systemic circulation. This can be attributed to the fact that DL has the capability to squeeze through pores one-tenth of its diameter and thus can penetrate the stratum corneum spontaneously, hence improving the transdermal delivery of an entrapped drug ,10. Due The surface modification of liposomes is one of the most promising ways to improve their stability. Some improvements have been attained in the chemical and physical stability of polymer-coated liposomes prepared with poloxamer, chitosan (CS), carboxymethyl chitosan, and dextran derivatives . CoatingMoreover, the use of CS can increase the efficacy of the loaded drug because it ensures a sustained release and improves the cellular uptake of the liposomes by cells owing to the chitosan positive charge. In addition, CS mucoadhesive properties will prolong the residence times at the site of drug absorption due to the increased contact of the carrier with the absorbing site. Previous studies have confirmed the important role of CS in the transdermal delivery system. Park SN. et al. observed that CS-coated liposomes had superior permeation compared to the uncoated sample . Yadav AThe interest of this work includes the association of the transdermal delivery of GLP and CS-DLs as novel carriers of delivering GLP. This approach could be an effective and noninvasive sustained-release formulation to achieve the hypoglycemic effect. Therefore, this study was designed to develop GLP-loaded DL containing two types of edge activators (Tween 80 and sodium deoxycholate) and coated DL with chitosan to obtain chitosomes. The prepared formulations were assessed in terms of particle size, zeta potential, polydispersity index, GLP entrapped efficiency, vesicle morphology, and in vitro GLP release, while stability and deformability studies were also carried out. Transdermal films of GLP were prepared and evaluated. Pharmacokinetics and pharmacodynamics studies were performed following transdermal application to adult male Wistar albino rats after the induction of diabetes using the Streptozotocin model.Glipizide was purchased from A.M.S.A. Anonima Materie Sintetiche and Afini . Lipoid S100 (phosphatidylcholine (PC), soybean lecithin, >94% PC) was obtained from Lipoid GmbH . Cholesterol and low-molecular-weight chitosan , degree of deacetylation (DD of 92%) were purchased from Sigma-Aldrich Chemical Co. Ltd. . Sodium deoxycholate, methanol, and acetonitrile (HPLC grade) were obtained from Fisher Scientific Co., . Tween 80 was obtained from BDH Organics . Hydroxypropyl methylcellulose (HPMC) low viscosity (viscosity of ~3 cP for 2% aqueous solution), MWT ~20,000 Da; (HPMC LV grade E3) was purchased from Dow Chemical Co. . Propylene glycol (PG) was obtained from WINLAB Laboratory chemical reagents . Streptozotocin and sodium carboxymethyl cellulose  were purchased from Sigma-Aldrich . All the other chemicals were of analytical grade.w/v). The thin-film hydration method was adapted to prepare the DL and CL, with slight modifications [Deformable liposomes (DL) were prepared using different molar ratios of lipoid S100 (PC), cholesterol, Tween 80, and sodium deoxycholate, as shown in ications ,21. BrieThe resultant dry film was hydrated with an appropriate volume of pre-warmed distilled water at 45 °C and vortexed for 30 min to form multilamellar vesicles (MLVs) of liposomes. The formed MLVs were then subjected to a probe sonication  for 10 min to achieve a vesicle with a nano-size range. Light centrifugation was employed at 5000 rpm for 5 min to remove the metals shaded from the probe sonicator.w/v) was obtained by dissolving CS in 0.5% (v/v) acetic acid solution  under magnetic stirring at room temperature for 5 h and filtered using a vacuum pump . The CS-coated liposomes were prepared by the dropwise addition of an equal volume of liposomes to specific concentrations of CS solutions (0.2% w/v) under magnetic stirring at room temperature for 30 min. Several chitosomes were obtained containing 0.1% w/v CS, as shown in w/v). The obtained formulations were stored in a refrigerator at 4 °C for further studies.To obtain CS-coated liposomes, chitosan (CS) stock solution  of the formulations were measured by dynamic light scattering using a Zetasizer Nano ZS . A clean cuvette was filled with a 1:100 solution of the formulations to filter deionized water, equilibrated at 25 °C, and then subjected to vortexing for 3 min. Zeta potential was determined by the Zetasizer Nano ZS  based on electrophoretic mobility after proper dilutions were performed. All the results are presented as the average of triplicate measurements.total). Secondly, a certain volume of each formulation was exposed to ultracentrifugation  at 4 °C for 45 min. After that, the resultant supernatant was dissolved in methanol; this represented an un-entrapped (Wfree) fraction. All the samples were filtered using a 0.45 μm syringe filter  before being analyzed by a reversed-phase HPLC method with slight modification [Entrapment efficiency (EE) is another parameter to investigate the efficiency of the obtained formulations. The EE% was established by the indirect method using the ultracentrifugation technique. Firstly, an accurate volume of each formulation was dissolved in methanol (1:10), and this represented the total amount of drug . The size of the vesicles was determined before and after the extrusion by dynamic light scattering using a Zetasizer Nano ZS. The deformability index The deformability of the liposomes was assessed using the extrusion method with slight modifications . BrieflyThe morphology of the selected CS-coated DL formulations was visualized using transmission electron microscopy (TEM). They were stained with 2% urany1 acetate solution, and the excess was removed using filter paper. The samples were allowed to air-dry in a dust-free environment and then observed by TEM  at 60 kV.v/v Tween 80 in PBS; pH 7.4) was determined to ensure the sink condition during the experiment. An artificial cellulose membrane  was mounted between the diffusion cells’ donor and receptor compartments. The receptor medium (12.5 mL) was composed of 0.5% (v/v) Tween 80 in PBS (pH 7.4) and adjusted at 37 °C. The formulations were added to the donor compartment, then at fixed time intervals  an aliquot of 1 mL was withdrawn from the receptor media and replenished with an equal volume of fresh receptor medium [w/v) in water (GLP control) for comparison. All experiments were performed in triplicate. A kinetic analysis of the in vitro release data was performed after fitting the obtained data to different kinetic models: zero-order, first-order, Higuchi, and Korsmeyer–Peppas.An in vitro drug release study was achieved using Franz diffusion cells . Initially, the saturated solubility of GLP in the receptor fluid  and PG (1% w/v) were dissolved in distilled water using a magnetic stirrer to prepare the casting solution. The casting solution was subjected to sonication for 1 h to remove air bubbles. Then, it was left overnight in the refrigerator at 4 °C to attain a clear solution. An accurate volume of each liposome formulation containing GLP equivalent to 0.5 mg/cm² was added to an exact volume of casting solution to provide a total volume equal to 1 mL/cm² under magnetic stirring for 15 min at room temperature. Sonication was performed for 30 min to remove the air bubbles produced by stirring. The solution was poured into a Teflon plate with a 6.3 cm diameter (about 31 cm² area) and kept in an oven at 40 °C until the complete evaporation of the water. After drying, the film was carefully peeled off from the Teflon plate and then cut into pieces of 1.6 cm diameter. The obtained films were stored in aluminum foil bags and placed in a desiccator for further experiments. For pure GLP transdermal film, the same amount of GLP as used in GLP DL and chitosomes was dispersed in the polymer solution. The same procedure as mentioned above was followed.All the prepared formulations were selected to be incorporated into transdermal films. The films were prepared using the solvent casting technique ,28. HPMCThe film’s thickness was measured using a screw gauge micrometer  at five different points on the film and the average values were calculated. The weight variation of the five films (2.01 cm²) was determined separately using a digital balance  and the weight average was calculated . MoisturThe drug content of the prepared films was evaluated. Films were cut into small pieces (about 2.5 cm²) and dissolved in a 100 mL mixture composed of phosphate-buffered saline pH 7.4 and methanol in a ratio of 50:50. The solution was shaken continuously in a shaking water bath  adjusted to 100 rpm at 25 °C for 24 h. Furthermore, to confirm the complete extraction of the drug from the film, the solution was sonicated for 1 h under stirring for 5 min. The solution was filtered using a 0.45 μm syringe filter  and analyzed using reversed-phase HPLC . The folv/v) Tween 80, and it was adjusted to 37 °C. The amounts of the GLP that permeated at several time points  were analyzed by HPLC. All samples were analyzed in triplicate and the permeation profiles were constructed by plotting the cumulative amount of GLP permeation versus time.The ex vivo experimental protocol was approved by the Research Centre Ethics of King Saud University, College of Pharmacy, Riyadh, Saudi Arabia (Ref. No.: KSU-SE-19-73). Male Wistar rats weighing 200–250 g were anesthetized and the abdominal skin’s hair was removed. Full-thickness abdominal skin was excised and the subcutaneous fat was carefully removed. The skin was washed with PBS and stored at −20 °C till further use ,32. An e2/h) through the rat skin were calculated from the slope of the linear portion of a plot of the cumulative amount that permeated through the rat skin per unit surface area versus time; the extrapolation of this line intercepted the x-axis at a time equal to the lag time (TL). The permeability coefficients  were obtained by dividing J by the initial drug concentration (C0) in the donor compartment.The cumulative amount of GLP that permeated through the skin membranes was plotted as a function of time. The slope and intercept of the linear portion of the plot were derived by regression. GLP fluxes . The films were sputter-coated with gold before scanning.Among the transdermal films that were prepared earlier, GLP-CS-DL1 and GLP-CS-DL3 were selected for further in vivo experiments based on their high cumulative amounts of drug permeation at the end of 24 h.Adult male Wistar albino rats weighing 200–250 g were used, and the in vivo experimental protocol was approved by the Research Centre Ethics of King Saud University, College of Pharmacy, Riyadh, Saudi Arabia (Ref. No.: KSU-SE-19-73). The animals were acclimated to the laboratory conditions for at least 1 week before the start of the experiments. The animals were kept in standardized conditions . They were housed in polypropylene cages at four per cage with free access to water and chow diet ad libitum.w/v of sodium carboxymethyl cellulose. The animals were treated with GLP-CS-DL1 transdermal film (group 2) and GLP-CS-DL3 transdermal film (group 3) using an equivalent of 5 mg/kg of GLP. Group 4 was treated with untreated GLP transdermal film (GLP-TDF) . Before applying the transdermal films, the skin was gently wiped with warm water and additionally secured by medical adhesive tape. The blood samples were withdrawn at different time intervals  through the tail vein using heparinized tubes. The plasma was separated by centrifugation and stored at −20 °C until further examination. A rat plasma sample of 0.1 mL and 0.1 mL of 0.1 N HCl was vortexed for 3 min, then 3 mL of acetonitrile was added for the precipitation of the plasma proteins. The mixture was shaken using a vortexer for 5 min followed by centrifugation for 10 min at 6000 rpm, and the precipitates were removed by a 0.22 μm syringe filter. The organic phase was evaporated under a nitrogen environment and the mobile phase was added to this residue followed by vortex mixing. Pharmacokinetic studies of GLP were performed using a reversed-phase HPLC method.Twenty overnight-fasted rats whose hair has been removed earlier were divided into four groups of five rats each. Group 1 was given oral GLP suspension (5 mg/kg) in 2.5% 1/2) was calculated from the terminal elimination rate constant using the formula t1/2 = 0.693/K. The peak plasma concentration (Cmax) and its corresponding time (Tmax) were read directly from the plasma drug concentration–time profile. The area under the curve for each drug concentration–time curve from 0 to 48 h AUC0−48 was calculated using the trapezoidal rule.The results obtained for the plasma concentration of GLP were presented as the mean ± S.D. A PK analysis was performed with the aid of Excel 2010 using the PK Solver Add-In software. The elimination rate constant (K) was estimated from the linear regression analysis of the terminal portion from the log-linear plasma concentration–time profile. The elimination half-life (tn = 5). Streptozotocin (55 mg/kg) solution was freshly prepared in 0.1 M citrate buffer at pH 4.5. The rats were fasted overnight before the induction of diabetes. The prepared streptozotocin solution was administered via intraperitoneal (i.p.) injection. A 5% (w/v) glucose solution was given to animals to overcome drug-induced hypoglycemia for the next 24 h. After 24 h, the fasting blood glucose level was measured to confirm type 2 diabetes. The blood glucose levels were determined using an Accutrend Alpha Glucometer . The degree of hyperglycemia was evaluated, and rats with blood glucose levels > 250 mg/dL were chosen to complete the study [The streptozotocin model was used to induce hyperglycemia in the diabetic rats . The values at Liposomes are one of the most successful carriers used for transdermal delivery, because of their interesting properties such as biocompatibility and FDA approval for human use. In addition to the drug protection capability of PCL from degradation, PCL NPs have the ability to offer sustained drug release. When CS is used to coat liposomes, significant structures are attained with improved drug skin permeability. Moreover, the surface modification of liposomes by CS might allow the nanocarriers to bind to the cellular membrane. The cost-effectiveness considers the treatment with liposomal formulations more expensive, more effective, and safer than conventional formulations, with the highest cost concerning the lipid used. Furthermore, the cost-effectiveness occurs from being able to take a lower dose for the same outcome. This recommendation should be followed to increase the efficacy of the delivery system while confirming its safety once inside the body, as well as the cost-effectiveness of nanocarriers in medicinal applications.All the liposomes investigated were in the nanometer range, as shown in Moreover, coating liposomes with CS is one of the approaches used to enhance their stability and increase the loaded drug’s efficacy, since it assures a sustained release . This bePDI is an indicator of particle homogeneity, and its value ranges from 0.0 to 1.0. As the value is close to zero, the vesicle’s homogeneity becomes higher . From TaZeta potential values give an indication of the surface electrical charge of the vesicles, which is a particularly significant parameter that affects liposome behavior . A high The zeta potential of the CS-coated liposomes was measured and is displayed in The EE% results  showed tThe EE% of GLP in different CS-coated liposomes formulations ranged from 51.90 ± 1.25% (GLP-CS-DL2) to 88.59 ± 0.37% (GLP-CS-DL1). These results demonstrated that the CS-coated liposomes had a decreased EE% compared to uncoated liposomes. The reduction in EE% observed in the CS-coated liposomes might be explained by the fact that CS is expected to be both vesicle entrapped and surface available . MoreoveThe deformability principle is based on the ability of the vesicles of liposomes to maintain their size after extrusion through a polycarbonate membrane, which has a certain pore size. The change in the vesicle size was expressed in terms of the deformability index (DI). The DI is defined as the degree to which the liposomes deform following extrusion and do not recover to their original size. The greater the DI, the less deformable the liposomes that were unable to regain their previously large size . The CL The transmission electron microscope (TEM) is a significant description method for the size and shape of liposomes. TEM images presented in It was reported that positive drug therapy can be controlled by the residence time of the nanocarriers in vivo. Therefore, an in vitro release study may imitate a vesicle’s behavior inside the body based on the residence time. The GLP release profiles from liposomes and chitosomes were evaluated using the Franz diffusion technique. The in vitro release of GLP through CS-coated liposomes was found to be slower than that through liposomes, which was thought to be due to the presence of CS. The release behavior of GLP from the CL-, DL-, and CS-coated CL/DL as a function of time is illustrated in p < 0.05). The liposomes showed an initial burst release of about 25.74 ± 5.61%, 26.43 ± 3.81%, 20.65 ± 1.58%, and 22.82 ± 4.71% of GLP from GLP-CL, GLP-DL1, GLP-DL2, and GLP-DL3, respectively, after 2 h, followed by a more gradual release for the following 24 h. The initial burst release might be due to the desorption of GLP from the liposomal surfaces.By using a GLP suspension (control), 5.55 ± 1.64% of the drug released after 2 h was detected. However, a significant increase was observed in the drug release up to 13.01 ± 1.59% after 24 h   than the other models in the case of uncoated liposomes. Hence, the drug release profile of GLP follows the diffusion mechanism. Furthermore, the release data of coated liposomes were fitted using Korsmeyer and Peppas’s well-known empirical equation. As shown, the n (release exponent) value is used to characterize different release mechanisms. It was found that n or the diffusion exponent was 0.5 < n < 1, which implies that the drug release from the system follows a non-Fickian transport pattern. In the non-Fickian pattern, the mechanism of drug release is governed by diffusion and swelling. The swelling of the CS coating occurs slowly, and the diffusion process simultaneously causes time-dependent anomalous effects.The in vitro drug release profiles were evaluated in different mathematical models, such as the zero-order, first-order, and Higuchi kinetics models, as shown in For safe and efficient use of liposomes, it is essential to establish their stability, which has considerable effects on liposome storage and further applications. A stability study was carried out for short time (28 days) for GLP-loaded CL, DL1, DL2, and DL3 formulations and CS-coated ones. The changes in particle size and PDI are shown in Thus the obtained film was formulated with GLP-loaded coated and uncoated liposomes with two goals, the controlled transdermal delivery and the highest percutaneous of GLP. In consideration of the treatment of hyperglycemia, GLP-loaded film should be of fixed dimensions with content uniformity to give sustained release. The results found that physicochemical characteristics, such as thickness, weight variation, moisture uptake, drug content, and folding endurance, exhibited a slight variation among the obtained films. These parameters evaluated for films are listed in To confirm the performance of the sustained release of GLP after transdermal application and observe its in vivo behavior, an ex vivo skin permeation experiment was employed. The experiment was carried out in vertical Franz’s diffusion cells. The permeation results of GLP from the selected different films through excised male Wistar rat skin are presented in 2, respectively. On the other hand, in CS-coated liposomes, GLP-CS-DL1-TDF and GLP-CS-DL3-TDF delivered 85.117 ± 5.004 and 57.273 ± 2.358 µg/cm2 of GLP, respectively, after 24 h. These data showed a sustained release behavior during the 24 h release study. The initial drug release usually occurs due to the fast release of the drug entrapped near the surface of films for which the film drying process was employed. The GLP entrapped deeper in the core matrix of the film takes a longer diffusion path to reach the surface. It has been stated that the use of HPMC in the preparation of transdermal film might be responsible for controlling the release of the drug [After 24 h, the cumulative amounts of GLP that permeated from GLP-DL1-TDF and GLP-DL3-TDF were 40.645 ± 1.139 and 45.834 ± 3.208 µg/cmthe drug . The addthe drug .Moreover, the amounts of GLP that permeated were also influenced by the type of edge activators contained in the DL. Moreover, GLP-DL1-TDF that contained Tween 80 resulted in a slightly lower permeation than the one consisting of sodium deoxycholate (GLP-DL3-TDF). This may be attributed to the surfactant’s hydrophilicity, which determined the diffusion rate through the hydrophilic matrix of the transdermal film. The existence of sodium deoxycholate, a hydrophilic surfactant with an HLB value equal to 16, in GLP-DL3-TDF, produced liposome vesicles with a total hydrophilicity higher than GLP-DL1-TDF that consisted of Tween 80 and HLB equal to 15. The presence of Tween 80, as the only surfactant utilized in GLP-DL1-TDF, led to a liposomal vesicle with a relatively lower hydrophilicity. This might interfere with the vesicle mobility within the polymer matrix, allowing a lower GLP delivery through the skin . TherefoFurthermore, the cationic nature of CS-coated liposomes also plays a significant role in improving the rate of GLP permeation. High amounts of GLP were detected for GLP-CS-DL1-TDF and GLP-CS-DL3-TDF. The mechanism for the enhancement of the GLP permeation across the rat skin membrane could be attributed to an electrostatic attraction that developed between the negatively charged skin surface and the positive charge of CS-coated liposomes. The positively charged liposomes have the potential to disrupt the negatively charged tight junctions in the skin. The bioadhesion force of CS could improve the vesicle’s contact period with the skin, causing higher penetration. Many studies have concluded that the vesicles’ charge has a significant impact on the permeation of the drugs through the skin. Positively charged vesicles improve the drug permeation more than negatively charged ones . Thus, t2) of the regression lines could be used to assess the stability of drug permeation. The better the stability of the steady-state flux (J) is, the closer the value of 2R is to 1. According to 2 values of GLP-CS-DL1-TDF and GLP-CS-DL3-TDF directly applied were both higher than 0.98, showing great stabilities of steady-state flux (J) of 8.436 ± 0.915 and 5.421 ± 0.829 μg/cm2/h, respectively. Furthermore, GLP-CS-DL1-TDF and GLP-CS-DL3-TDF showed shorter lag times of 2.165 ± 0.136 and 3.778 ± 0.341 h, respectively. The transdermal J of GLP from the GLP-DL1-TDF and GLP-DL3-TDF were 2.725 ± 0.071 and 4.322 ± 0.798 μg/cm2/h, respectively. The lag time was 7.092 ± 1.233 and 4.043 ± 0.838 h for GLP-DL1-TDF and GLP-DL3-TDF, respectively, which was larger than that of the other coated DL transdermal film. Due to its higher release rate, the lag time was shorter as well. However, the difference in the cumulative permeation parameters of films suggested the critical role of the formulation composition, resulting in diverse skin permeation profiles. The increased positivity of the formulation could increase the skin permeation of GLP from the corresponding transdermal film. This might be clarified by the fact that CS coating might facilitate interaction with the stratum corneum (the main barrier in drug skin transport). Since GLP-CS-DL1-TDF and GLP-CS-DL3-TDF have proved their transdermal ability in ex vivo studies, in vivo experiments (pharmacokinetic and blood glucose level tests) were conducted to further investigate their in vivo bioavailability and hypoglycemic consequences.The skin permeation parameters for GLP-DL1-TDF, GLP-CS-DL1-TDF, GLP-DL3-TDF, and GLP-CS-DL3-TDF directly applied were calculated and are listed in The SEM of the selected transdermal films  showed tmax (13.63 ± 1.18 μg/mL) at a Tmax of 2.00 ± 0.49 h after oral administration, followed by a rapid decrease in GLP at 24 h and 48 h. In the case of the transdermal film containing GLP (GLP-TDF), the values of Cmax and Tmax were 2.42 ± 0.47 μg/mL and 12±00 h, respectively. These values were significantly different compared to those found for the oral route of GLP (p < 0.05). While transdermal film containing GLP-CS-DL1-TDF showed a Cmax of 7.87 ± 1.06 μg/mL and a Tmax of 12.00 h, GLP-CS-DL3-TDF showed a Cmax of 4.82 ± 1.03 μg/mL and Tmax of 12.00 h. These values were significantly different compared to those found for the oral route of GLP (p < 0.05). Measurable concentrations of GLP were detected within an hour after the application of the transdermal film and a relatively low Cmax and prolonged Tmax (12 h) were observed over 48 h. This behavior of transdermal film related to oral administration could be attributed to the effective barrier of the skin. This could be also due to the quick absorption of drugs via the oral route, with drugs administered through the transdermal route permeating slowly with continuous absorption. Furthermore, the biological half-life (t1/2) of GLP after transdermal application was prolonged to about 7.96 ± 0.23–9.73 ± 0.47 h compared to 2.25±0.61 h after oral administration (p < 0.05).The plasma concentrations of GLP formulations after oral and transdermal administration are shown in 1/2 values and increased mean residential time (MRT) values. The MRT of GLP was also prolonged from 4.96 ± 0.55 h  to 27.09 ± 1.08 h (GLP-CS-DL1-TDF). Therefore, the transdermal film could accomplish sustained release and preserve the drug in the body for a longer period. The values of AUC of the transdermal films (GLP-CS-DL1-TDF and GLP-CS-DL3-TDF) were significantly high in comparison with those achieved through oral administration. This effect was attributed to the maintenance of the concentration of GLP for an extended time from the transdermal films. The detected high values of AUC from the transdermal delivery system compared to the oral route indicate the increased bioavailability of GLP in rats. This could be due to the slow and continuous delivery of GLP to the systemic circulation, which may lead to daily glycemic control being achieved in diabetic subjects.The pharmacokinetic profile of GLP when delivered as a transdermal film showed a prolonged release behavior. During the first 12 h, the transdermal film delivered quite a lot of its drug content, followed by an extended release of the drug in the plasma until 48 h. This performance was supported by high tThe observation of the blood glucose level was considered to estimate the in vivo performance of the hypoglycemic agent, GLP. The change in the blood glucose level–time profile of the five groups is displayed in The glucose level reduction at 8 h was high with oral administration compared to all other formulations. Moreover, at 48 h, the hypoglycemic effect declined and the blood glucose levels were increased. For GLP-TDF, the decrease in the blood glucose level was monitored for up to 8 h . The tramax and higher AUC that could improve the therapeutic efficiency of GLP. Moreover, a higher hypoglycemic effect was obtained after the topical application of the selected formulation of GLP-CS-DL1. Chitosan-coated DL-based transdermal films may prove to be a suitable nano-carrier for percutaneous penetration of GLP. It improves its transport across the skin’s stratum corneum, thus improving its bioavailability.Different types of DLs were successfully prepared using the thin-film hydration method and the prepared DLs were coated with CS. All the DLs and CS-coated DLs were investigated in terms of their physicochemical parameters and in vitro release. All the liposomes investigated were in the nanometer range with homogenous size and distribution (PDI ≤ 0.5) and sufficient high zeta potential values to provide good stability for the vesicles. Moreover, DLs showed superiority in obtaining high entrapment efficiency over the conventional ones. Because of DLs deformability, the release of loaded GLP through the membrane in the in vitro study increased from the DLs compared to the conventional liposomes. On the other hand, an in vitro release study confirmed that the DLs could reach higher cumulative release amounts of GLP compared to the CS-coated DLs. In addition, the transdermal films containing DLs and CS-coated DLs were prepared with efficient physiochemical properties. An ex vivo skin permeation study demonstrated the sustained release of GLP from the transdermal films, which might allow a more stable plasma level of GLP to be reached over time. CS-coated DLs, such as GLP-CS-DL1 or GLP-CS-DL3, showed a high amount of permeated GLP. GLP-CS-DL1 showed comparable pharmacokinetic parameters to those of oral GLP, but with a much lower C


Aim: To evaluate the accuracy of two PCR-based techniques for detecting SARS-CoV-2 variant Alpha (B.1.1.7). Materials & methods: A multicenter prospective cohort with 1137 positive specimens from Slovenia was studied. A mutation-based assay (rTEST-COVID-19 qPCR B.1.1.7 assay) and amplification curve pattern analysis of the Allplex SARS-CoV-2 assay were compared with whole-genome sequencing. Results: SARS-CoV-2 variant Alpha was detected in 155 samples (13.6%). Sensitivity and specificity were 98.1 and 98.0%, respectively, for the rTEST-COVID-19 qPCR B.1.1.7 assay and 97.4 and 97.5%, respectively, for amplification curve pattern analysis. Conclusion: The good analytical performance of both methods was confirmed for the preliminary identification of SARS-CoV-2 variant Alpha. This cost-effective principle for screening SARS-CoV-2 populations is also applicable to other emerging variants and may help to conserve some whole-genome sequencing resources. Since the beginning of the COVID-19 pandemic, more than 1500 variants of the SARS-CoV-2 virus have been characterized. The accumulation of single or multiple mutations by the variant can affect the phenotypic presentation of the variant and alter its transmissibility, infectivity, virulence or antigenicity . It has SARS-CoV-2 variant Alpha (B.1.1.7) spread rapidly in the UK in autumn 2020 and quickly became the most prevalent variant in other parts of the world, with a global prevalence of up to 46% . CurrentThe ideal method for genomic surveillance of SARS-CoV-2 would need to detect all critical mutations and confirm the variant, preferably simultaneously with baseline diagnostics. It would also need to be technically and financially accessible to a sufficient number of laboratories. Genomic changes in a variant virus can alter the detection kinetics of a specific target on RT-PCR (reverse transcription PCR) assays, which also alters the pattern of the amplification curve, which is an assay-specific characteristic . These cIn Slovenia, systematic non targeted and targeted genomic surveillance of SARS-CoV-2 was introduced in January 2021 by combining WGS of 10% of all PCR-positive samples and screening of another 10% of PCR-positive samples by specific real-time RT-PCR to detect variant Alpha, respectively. With the increasing number of PCR-positive samples during the third wave of the epidemic and the increasing prevalence of variant Alpha, it became necessary to modify the surveillance protocol. The primary aim of the study was to verify whether the specific real-time RT-PCR used in the targeted approach can be used as a confirmatory test for variant Alpha, as claimed by the manufacturer. At the same time, we evaluated the accuracy of amplification curve pattern analysis of the Allplex SARS-CoV-2 assay as a complementary tool for rapid presumptive identification of variant Alpha in the four laboratories using this assay as the primary diagnostic method for SARS-CoV-2 detection.A total of 1139 routine samples positive in the Allplex SARS-CoV-2 assay  collected from 1 January to 31 March 2021, were included in the genomic surveillance protocol and analyzed in parallel using the rTEST COVID-19 qPCR B.1.1.7 assay  and WGS. For WGS extracted viral RNA from the positive samples was stored at -80 °C until transport and processed in batches once a week by the core laboratory. The study was approved by the institutional review board. The principles outlined in the Declaration of Helsinki were followed for the study and no additional samples were collected for the purpose of the study.The Allplex SARS-CoV-2 assay  was used as a routine diagnostic RT-qPCR in four clinical microbiology laboratories of the National Laboratory for Health, Environment and Food (NLZOH). In this assay, four sets of primers are used to detect the presence of the E, N, S and RdRP genes of SARS-CoV-2, with the latter two detected on the same fluorescent channel. The results are automatically interpreted by the supplied software Seegene Viewer , while the amplification curves can be additionally analyzed visually simultaneously or retrospectively. The assay was performed according to the manufacturer's instructions.The rTEST COVID-19 qPCR kit was used to detect the B.1.1.7 variant. The assay aims to detects two of the NTD deletions, ΔH69–V70 and ΔY144, characteristic of the B.1.1.7 variant. When these deletions are not present, amplification of the specific B.1.1.7 target versus the consensus target of the rTEST COVID-19 qPCR assay is delayed by at least five cycles, as a recommended cut-off by the manufacturer.The shapes and relative positions of the amplification curves of the E and S/RdRP genes for each sample were subsequently checked in the software Seegene Viewer. The curves were visually analyzed by four different operators, one per laboratory, and classified into three different patterns according to the criteria shown in Samples were obtained as RNA extracts and stored at -80 °C until further processing. Libraries were prepared with amplification enrichment using the ARTIC v3 kit according to protocol V.4 with recommended reagents and consumables. Finally, samples were sequenced on either the Illumina MiSeq or NovaSeq platform depending on availability. Consensus genome sequences were obtained by: mapping fastq reads to the reference genome (NC_045512) using BWA aligner (v 0.7.17); 2) implementing the ivar (v 1.3)/samtools  workflow to filter the quality of aligned reads, remove primer sites and generate the consensus sequence. PANGO lines were assigned using the command-line tool PLEARN (v1.2.13), which allows a maximum of 10% Ns.To evaluate the diagnostic performance of both the rTEST COVID-19 qPCR B.1.1.7 assay and the Allplex SARS-CoV-2 amplification curve pattern analysis in detecting variant Alpha, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value for each method were calculated using WGS as a reference. A receiver operating characteristic curve was generated to verify the cut-off value set by the manufacturer of the rTEST qPCR COVID-19 kit. Analysis in GraphPad Prism  resulted in a table of possible cut-off values and their respective sensitivities and specificities with 95% CIs compared with WGS. The cut-off value with the highest Youden’s index (sensitivity + specificity - 1) was considered optimal. A p-value of 0.05 was considered statistically significant. Predictive values were determined using GraphPad QuickCalcs . Agreement of results between amplification curve pattern analysis and WGS was assessed using Cohen’s kappa statistics with the following interpretive guidelines: no agreement (0.00), low agreement (0.00–0.20), fair agreement (0.21–0.40), moderate agreement (0.41–0.60), substantial agreement (0.61–0.80) and near perfect agreement (0.81–1.00). The analysis was performed using GraphPad QuickCalcs.A total of 17 variants of SARS-CoV-2 were characterized among 1139 RT-PCR-positive samples . VariantSupplementary Material. This value represents the maximum number cycles for which the amplification of the B.1.1.7 target on the S gene is delayed compared with the consensus target in a variant Alpha sample. The area under the curve was 0.9833 . The cut-off of 5.1 cycles was selected and the evaluation of the performance of rTEST qPCR assay was based on this cut-off value.The receiver operating characteristic curve to determine the optimal cut-off value of the rTEST qPCR assay is shown in the The sensitivity, specificity, PPV and negative predictive value using WGS as reference are shown in The agreement of the results between the amplification curve pattern analysis and the WGS was satisfactory when the results of all participating NLZOH laboratories were considered .For rapid determination of the specific SARS-CoV-2 variant, several screening protocols can be used based on the detection of nonspecific changes in diagnostic PCR target signals or on the detection of specific mutations associated with a particular viral variant. Failed positive spike gene signals have been evaluated and used in several countries facing rapid spread of variant Alpha (B.1.1.7) ,13,15,16Although the two most common lineages present in our sample B.1.258.17 and B.1.1.7 share one of the two NTD deletions that the rTEST q PCR COVID-19 B.1.1.7 kit is designed to detect, namely ΔH69-V70, we have shown that the assay is still able to distinguish between these variants and detect B.1.1.7 with 98% specificity at the selected cut-off value. To our knowledge, no other independent evaluation of this assay has been published to date.As additional variants of concern and variants currently under investigation have been introduced in Slovenia in the last 6 months, screening for only one of these variants was no longer a sufficient approach. The observation that the curve of the S/RdRp genes deviated from the expected shape of the curve, leading to possible prescreening of variant Alpha, was reported in the technical report of the manufacturer of the Allplex SARS-CoV-2 assay in February 2021 and later published by Italian authors . In onlyIn addition to the previously described delay in S/RdRP amplification, we found linearization of the S/RdRP gene amplification curve to be a separate and equally important change. Although, the interpretation criteria were descriptively defined, the combination of both patterns proved to be 97% sensitive and specific compared with WGS. Different analysts independently obtained satisfactory agreement of results with WGS in four different laboratories.Finally, one of the advantages of systematic genomic monitoring performed using WGS is also the ability to compare other laboratory methods and procedures to the gold standard. High-quality evaluation of the analytical performance of diagnostic tests and procedures allows laboratories to adjust their surveillance and diagnostic protocols and respond in a timely manner to new epidemiological circumstances.There are several limitations in our study. First, we were unable to determine the underlying molecular mechanism for the change in amplification curves; it is most likely due to a lower efficiency of the PCR reaction. Second, the specificity of such a nonspecific screening necessarily depends on the biological background of variants in a given geographic area, and cannot necessarily be extrapolated to other geographic locations. Third, following the rapid evolution of SARS-CoV-2 variants, the clinical utility of our results may be limited to areas where variant Alpha is still present, and to laboratories using a particular diagnostic assay that was also used in our situation. Fourth, there were some differences between sites in kappa. This can be explained by geographic variations of the virus, possible coinfection with different variants or by the subjective nature of the pattern analysis itself. Nevertheless, we report here the largest evaluation of its kind with more than 1000 SARS-CoV-2 genomes completely sequenced.We describe here a good analytical performance of the two methods for the preliminary identification of SARS-CoV-2 variant Alpha. They can both be used as screening tests and even as confirmatory tests in areas with high prevalence of this lineage. Considering the still high prevalence of this variant in several regions and an increase in the prevalence of other variants of concern, similar approach can be used to monitor the prevalence of known SARS-CoV-2 variants in a cost-effective manner and save some WGS resources.Genomic surveillance of the SARS-CoV-2 pandemic is important for planning, implementation and analysis of containment measures. Cheaper and faster approaches are needed for surveillance, especially in resource-limited settings. Simultaneous detection of the virus and typical variants is a promising and feasible approach, considering that the major circulating variants in a geographical area are known.The accuracy of two PCR-based techniques for the detection of variant Alpha (B.1.1.7) in a large multicenter prospective cohort in Slovenia from January to March 2021 with 1137 positive samples were evaluated.A specific mutation-based assay (rTEST COVID-19 qPCR B.1.1.7 assay) and nonspecific curve pattern analysis of the Allplex SARS-CoV-2 assay were compared with whole-genome sequencing.Variant Alpha was detected in 155 samples (13.6%). Sensitivity and specificity were high for both assays, 98.1 and 98.0% for the specific assay and 97.4 and 97.5% for the nonspecific analysis, respectively.The positive predictive value of both tests was 97.4 and 97.7%, respectively, with a prevalence of 49.5%.A good analytical performance of both methods was confirmed for the preliminary identification of SARS-CoV-2 variant Alpha and both can be used as screening and even as confirmatory tests in areas with high prevalence of this lineage.This principle of cost-effective way to screen SARS-CoV-2 populations is also applicable to other emerging variants and may help to conserve some whole-genome sequencing resources.Click here for additional data file.


The purpose was to assess the efficacy of 0.4% hyaluronic acid and 0.2% galacto-xyloglucan on the subjective symptoms of dry eye disease and invasive and non-invasive tear film signs in oral isotretinoin for acne vulgaris treatment. A prospective, longitudinal, single-blind, clinical study was performed in oral isotretinoin for the acne vulgaris consumer population. Subjective dry eye disease questionnaires and invasive and non-invasive tear film assessments were reported prior to and after 6 weeks of hyaluronic acid with galacto-xyloglucan (HA-GX) treatment vs. hyaluronic acid alone (HA). Participants in the HA-GX group reported a higher decrease in the ocular surface disease index (17.01 ± 11.36 score points) compared to the variation in participants in the HA group (11.61 ± 11.18 score points). Standard patient evaluation of eye dryness also decreased more in participants in the HA-GX group (4.06 ± 5.50 score points) than in participants who received HA alone (0.70 ± 3.16). Regarding non-invasive break-up time (NIBUT), participants in the HA-GX group first NIBUT achieved an increase of 1.75 ± 1.16 s while participants in the HA-alone group demonstrated an increase of only 0.54 ± 1.01 s. The HA-GX group mean NIBUT increased by of 3.72 ± 5.69 s; however, the value for the HA-alone group was 2.19 ± 5.26 s. Hyaluronic acid in combination with galacto-xyloglucan significantly decreased limbal and bulbar conjunctival redness classification and SPEED test outcomes. The inclusion of galacto-xyloglucan also increased BUT and mean NIBUT values compared to those obtained with hyaluronic acid alone. Dry eye disease (DED) is a multifactorial disease that affects the ocular surface, produces diverse symptoms and, in some cases, produces lesions that affect the anterior surface of the eye. It becomes more common with age and is more common in women than in men , 2. DED Artificial tears are used in the treatment of dry eye in these patients to relieve symptoms and signs. Normally, all artificial tears have an aqueous base to which different molecules are added that improves lubrication, viscosity, osmolarity, tolerance, and residence time on the ocular surface . HyaluroThe purpose of our research was to assess the effect of 0.4% non-crosslinked hyaluronic acid and 0.2% galacto-xyloglucan on tear film stability and to evaluate DED signs and symptoms in subjects treated with isotretinoin.We conducted this prospective, longitudinal, single-blind, single-center study at the Optics and Optometry cabinets of the Pharmacy School . This research was conducted according to the Helsinki Declaration and the Ethical Committee Board of the University of Seville.All the included subjects read and sign the informed consent form. An information sheet was provided to all subjects that provided details about the study procedure. The inclusion criteria were as follows: (1) Users with active isotretinoin treatment for at least the last 2 years, (2) age between 18 and 30 years old, (3) standard patient evaluation eye disease score above 0 points, (4) invasive break-up time (BUT) under 25 s, (5) completion of all examination procedures, and (6) comprehension of the aims of this research study in its entirety and signed an informed consent form before the measurements. The exclusion criteria were as follows: (1) any previous eye surgery, (2) any systemic diseases, and (3) contact lens use.® . The OSA allows a full assessment of the ocular surface through a combination of dry eye disease diagnostic tests. The instrument was placed in the slit lamp tonometer hall. Within the technical data, the image resolution was 6 megapixels; the acquisition mode was multishot and movie acquisition; the focus could be manual or automatic; Placido disc and NIBUT grids were available, colored, and sensitive to infrared cameras; and the light source was infrared LED or blue and with LED. Two subjective dry eye disease questionnaires were used: the Ocular Surface Disease Index (OSDI) and the Standard Patient Evaluation of Eye Dryness (SPEED) test.Non-invasive tear film analysis was performed with the Integrated Clinical Platform (ICP) Ocular Surface Analyzer (OSA) from SBM System®, distributed by ESTEVE Pharmaceuticals®, Barcelona, Spain, and manufacturer by Omisan Farmaceuti®, Guidonia Montecelio, Italy). This eyedrop was packaged in a multidose 10-milliliter bottle. Within the control group, eyedrop B  was 0.40% hyaluronic acid sodium salt and distilled water with ginkgo biloba, cranberry, fennel and spark asiatica, boric acid, sodium tetraborate, and sodium chloride with a sufficient quantity for 100 milliliters . This eyedrop was packaged in a multidose 10-milliliter bottle.Regarding the lubricants studied, eyedrop A  was 0.40% hyaluronic acid sodium salt, 0.20% galacto-xyloglucan (extracted from tamarind seed), mannitol, trisodium citrate dihydrate, citric acid monohydrate and isotonic buffered solution with a sufficient quantity for 100 milliliters . The degree of MGD was measured by the ImageJ method defined by Pult and Nichols  that detected the blood vessel fluidity of the conjunctiva, evaluating the redness degree with the Efron Scale . [2] Lipid layer thickness (LLT) evaluation with optic interferometry, evaluating the quantity of lipids layer into 7 different pattern categories . [3] Tear meniscus height (TMH) measurement evaluates the aqueous layer and is quantified within a millimeter caliper . [4] First and mean non-invasive break-up time  were evaluated with a special grid cone, which evaluates the quality of the mucin layer in seconds . To evaluate the meibomian glands, infrared meibography was performed with a COBRA Nichols .In a second phase, the subjects were re-evaluated after 6 weeks with a 12-h posology to quantify the ocular surface parameters and subjective questionnaires. Finally, after a rest period of 30 min, an invasive tear film examination was performed within the fluorescein break-up time test. The temperature and humidity room examination conditions were stable during all measurements.U-test. The correlation study was evaluated with the Spearman Rho test. For all tests, the level of significance was established at 95% . The sample size was evaluated with the GRANMO calculator . The two-sided test was used. The risk of alpha and beta was set at 5 and 20%, respectively. The estimated standard deviation (SD) of the differences was set at 2.06 [based on De-Hita-Cantalejo et al. . Descriptive analysis was performed with the mean ± standard deviation (SD) and . The normality distribution of the data was assessed with the Shapiro–Wilk test. Differences in qualitative variables were assessed with the chi-square test. The differences among the first, second and third OSA measurements were assessed with the Wilcoxon test. Differences within both eyedrop groups were analyzed with the Mann–Whitney o et al.  SD of thFifty eyes from 25 patients were included in this study. Twenty-five right eyes and 25 left eyes were included. Seven males and eighteen females were enrolled in this research. Demographic data about age, sphere refraction, cylinder refraction, axis refraction, CDVA (log MAR) and superior and inferior meibomian gland dysfunction percentages are presented in P = 0.18) for participants in the HA-alone group. Lipid layer interferometry for participants in the HA-GX group changed only 0.11 ± 1.39 degrees on the Guillon Pattern, similar to that for participants in the HA-alone group, which changed 0.12 ± 0.94 degrees on the Guillon Pattern (P = 0.38). The TMH for participants in the HA-GX group remained uniform, with a change of 0.009 ± 0.06 millimeters, similar to that for participants in the HA-alone group, which changed by 0.004 ± 0.03 (P = 0.60). Finally, regarding the first and mean NIBUT, participants in the HA-GX group achieved an increase of 0.57 ± 2.89 s in the FNIBUT; however, participants in the HA-alone group achieved a decrease of 0.64 ± 1.82 s (P = 0.79). In addition, the participants in HA-GX group reported an increase of 1.11 ± 5.10 s in the MNIBUT, while participants in the HA-alone group showed a decrease of 1.82 ± 11.16 s (P = 0.46).Limbal and bulbar conjunctival redness, lipid layer interferometry, tear meniscus height assessment, and first and mean NIBUT differences before and after treatment are presented in P = 0.59). OSDI and SPEED differences before and after treatment are presented in P = 0.06). According to the results of the SPEED test, the participants in the HA-GX group achieved a decrease of 1.23 ± 3.49 points, and the HA-alone group achieved a decrease of 3.00 ± 3.65 points (P = 0.93). A statistically significant difference before and after eyedrop treatment box and blot graph is presented in BUT test differences before and after eyedrop treatment are presented in The results of our research show an improvement in both patient-reported symptomatology and tear break-up time in subjects treated with oral isotretinoin after using artificial tears composed of 0.4% HA and GX at 0.2%. The heterogeneous nature of DED and the variability of signs and symptoms do not allow an accurate diagnosis. There is evidence of the influence of the drug at the ocular level, negatively affecting the meibomian glands and goblet cells and, as a consequence, altering the lipid and mucin layers of the tear , 8 and aP < 0.01). Molina-Solana et al.  . We also measured BUT invasively with fluorescein. The results showed a statistically significant increase in participants in the HA-GX group (8.69 ± 4.75 s – 11.35 ± 4.71 s) (P < 0.01). There were similarities between the structure of GX, tamarind seed polysaccharide (TSP) and the mucin MUC1 present in the epithelium of the cornea and conjunctiva  and SPEED (score), to classify the degree of dry eye according to its symptoms . In a loRegarding strengths and limitations, to the best of our knowledge, this clinical study demonstrated the efficacy of two types of eyedrops in oral isotretinoin for acne vulgaris for the first time. In addition, non-invasive ocular surface analyzer measurements were used. Within the limitations, the sample size and follow-up of the research could be improved to confirm these results. Furthermore, a double-blind design should reduce patient bias. Future lines of research should include the isotretinoin dose analysis and correlate it with the ocular surface signs and symptoms.Within the future research lines, eyedrop manufacturer laboratories should open the option of producing personalized lubricants for each patient. Regarding dry eye disease pathophysiology, one or more tear layers will be affected, so a different excipient in the eyedrop composition is needed. Therefore, the indication for dry eye disease treatment should include a thorough dry eye examination and evaluation of the causes that instigated it.In conclusion, hyaluronic acid in combination with galacto-xyloglucan significantly decreased limbal and bulbar conjunctival redness and SPEED subjective dry eye disease symptoms. Galacto-xyloglucan also increased the BUT and mean NIBUT compared to hyaluronic acid alone.The data presented in this study are available on request from the corresponding author. The data are not publicly available due to their containing information that could compromise the privacy of research participants.The studies involving human participants were reviewed and approved by the Ethical Committee Board of the University of Seville. The patients/participants provided their written informed consent to participate in this study.MS-G, CD-H-C, and J-MS-G: conceptualization and methodology. MS-G, CD-H-C, CM-L, and J-MS-G: writing—original draft preparation, writing—review and editing, and supervision. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Lung cancer is the major cause of cancer-related deaths around the world. Lung adenocarcinoma (LUAD), the most common subtype of lung cancer, contributed to the majority of mortalities and showed different clinical outcomes in prognosis. Tumor-infiltrated immune cells at the tumor site are associated with better survival and immunotherapy response. Thus, it is essential to further investigate the molecular mechanisms and new prognostic biomarkers of lung adenocarcinoma development and progression. In this study, a six-gene signature  was established to predict the prognosis of LUAD patients, as well as predictive value. The prognostic risk model was also significantly associated with the infiltration of immune cells in LUAD microenvironments. To sum up, a novel immune-related six-gene signature  was identified that could predict LUAD survival and is highly related to B cells and dendritic cells, which may provide a theoretical basis of personalized treatment for targeted immunotherapy. Lung cancer is currently the most aggressive cancer in terms of morbidity and mortality worldwide ,2,3. TheIt is known that a tumor immune microenvironment plays significant roles in tumor development and progression ,10,11,12Although many immune-related gene-based markers for LUAD are available, immunotherapy could only benefit a small proportion of patients ,20. Thern = 175) and stage T2-T4 (n = 357) from a TCGA dataset were assessed by using R package “DESeq2 (v 1.26.0)” with the criteria of |log2 (Fold change)| > 1 and p.adj < 0.05 [https://www.immport.org/home) [Differentially expressed genes (DEGs) between LUAD tumor samples at stage T1 (j < 0.05 . The volrg/home) . The difThe 40 DEIRGs were obtained from the intersection between DEGs and IRGs. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using clusterProfiler (v3.14.3) and org.Hs.eg.db (3.10.0) . The bubDifferentially expressed immune-related genes (DEIRGs) were used for the Least Absolute Shrinkage and Selection Operator (LASSO) coefficient screen using the R software glmnet (v4.1-2) and survival (3.2-10). The prognostic immune-related gene signature (risk model) with coefficients were selected based on the optimal lambda value. Overall survival (OS), disease specific survival (DSS) and the progression-free interval (PFI) between high and low risk score groups were conducted using the R software survminer (0.4.9) and survival (3.2-10) . UnivariThe ssGSEA algorithm was applied for the correlation between the risk score and 24 tumor-infiltrated immune cells by using GSVA (1.34.0) ,27. The https://www.proteinatlas.org). The Genetic alteration analysis was performed using the cBioPortal for Cancer Genomics  [The immunohistochemistry images of six gene signatures in LUAD tumor tissues and normal tissues were obtained from the Human Protein Atlas (HPA) database  .https://www.procell.com.cn). The A549 cells were cultured in Ham’s F-12K Medium . The NCI-H1975 cells were cultured in RPMI1640 medium . The Calu-3 cells were cultured in modified Eagle’s medium . The BEAS-2B cells were cultured in Dulbecco’s modified Eagle’s medium . All cells were cultured in the specific mediums supplemented with 10% fetal bovine serum , penicillin sodium (100 U/mL) and streptomycin (100 mg/mL). The cells were incubated in the thermostatic cell incubator at 37 °C with 5% CO2. Total RNA was extracted using the RNeasy Plus Mini Kit . Quantitative PCR (qPCR) was performed using the ABI 7900 qPCR system. Relative RNA expression was normalized to GAPDH and calculated by the 2−ΔΔCt method. The primers were listed in Human lung cancer cell lines  and human normal epithelial cells BEAS-2B were purchased from Procell  and the qPCR analysis was performed by GraphPad Prism. DEGs were analyzed by “ggplot2”. GO-KEGG and GSEA analyses were performed by “clusterProfiler”. The LASSO analysis was conducted by “glmnet and survival”. Univariate and multivariate Cox regression analyses were assessed by using the “survival”. The Kaplan–Meier analysis was performed using the “survival” and “survminer” R packages. An immune cell infiltration analysis was applied by “GSVA”. A The workflow of this study is illustrated in We used a LASSO Cox regression model to perform the overall survival analysis of 40 immune-related potential prognostic genes A. Six geThe risk score of each LUAD patient was a linear combination of each six-gene signature expression and its risk coefficient. High- and low-risk groups of LUAD patients were divided according to the median risk score. The distribution plots of risk scores and the outcome status, including the Overall Survival (OS), Disease Specific Survival (DSS), and Progression-Free Interval (PFI) of the gene signature, are shown in We performed the time-dependent receiver operating characteristic (ROC) plotter and found that the values of the area under the curve (AUC) for the six-gene signature risk score at 1, 3, 5, and 10 years were all above 0.6 A. Next, To further understand the biological functions of the six-gene signature risk score in LUAD, we performed enrichment analyses of GO and KEGG pathways. Results showed that the six-gene signature was enriched in lymphocyte mediated immunity, leukocyte proliferation, mononuclear cell proliferation, and lymphocyte proliferation A. Consisp < 0.01.Next, we investigated the possible relationship between multiple clinicopathological features and the six-gene signature risk score. The correlation analysis indicated that a higher risk score was significantly associated with age, gender, pathologic stages, T/N/M stages, and OS/DSS/PFI events A–I. Morep < 0.05), whereas T gamma delta (Tgd) and Th2 cells were highly expressed in the high-risk group. Furthermore, we investigated the correlation with tumor lymph cells by Spearman’s correlation analysis. The lollipop chart indicated that risk score was positively related to Th2 cells and Tgd (To further explore the association between the six-gene signature risk score and immunity, we firstly evaluated the correlation with 24 types of tumor-infiltrating immune cells in the high- or low-risk groups using the ssGSEA algorithm A. Result and Tgd B. Moreov and Tgd B.p < 0.05, In addition, we also investigated the prognostic significance of 22 types of tumor-infiltrating immune cells. The Kaplan–Meier analysis demonstrated that the lower infiltrating abundance of B cells and dendritic cells were significantly associated with better OS (We also evaluated the association between the six-gene signature and tumor immune infiltration to calculate the immune score, ESTIMATE score, and stromal score using the ESTIMATE package. We observed that there was a significant difference in the immune score and stromal score between the low-risk group and the high-risk group, except for the ESTIMATE score A–C, suggThe mRNA level of CR2, INSL4, FGF5, RAET1L and TNFRSF13B was upregulated in LUAD tumor tissues compared with paired normal tissues whereas AGER was downregulated A. The prIn recent years, the development of cancer treatment strategies has been toward immunotherapy ,31. CellNowadays, it has become a growing focus for research to construct prognostic models based on lncRNA, miRNA, and mRNA to predict the prognostic or diagnostic value in tumors . In thisStudies have shown that a tumor microenvironment plays a key role in NSCLC development and is closely related to immunotherapy response ,44. GiveThe present study identified an original immune-related risk model for LUAD. There are some highlights in this study. First, we did a comprehensive analysis of the six-gene signature in LUAD, including the functional enrichment, the correlation with clinical features, the tumor-infiltrated immune cells, the immune checkpoints, and the expression profiles in tissues and cell lines. Second, a nomogram and calibration were used to further validate the accuracy for clinical outcomes for the risk model in LUAD. Nevertheless, there are some limitations in this study. First, clinical LUAD samples need to be collected to verify the validity and accuracy of the prognostic model. Second, relevant functional and mechanical experiments should be carried out to further explore the effect of this risk model in vitro and in vivo.In conclusion, we established an immune-related prognostic gene signature which was significantly associated with the tumor immune microenvironment. The six-gene signature model could predict the prognosis and survival of LUAD patients, as well as imply a better response to immune-based therapies, which may provide a theoretical basis for the prognosis and immunotherapy of LUAD.